Molecular markers associated with mal de rio cuarto virus in maize

ABSTRACT

This invention relates to methods for identifying maize plants that having increased MRCV resistance. The methods use molecular markers to identify and to select plants with increased MRCV resistance. Maize plants generated by the methods of the invention are also a feature of the invention.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.14/585,012 filed on Dec. 29, 2014, which claims the benefit of U.S.Provisional Patent Application Ser. No. 61/920,859, filed Dec. 26, 2013,the disclosures of which are hereby incorporated herein in theirentirety by this reference.

FIELD OF THE INVENTION

The present invention relates to methods useful in selecting forincreased Mal de Rio Cuarto Virus resistance in maize plants.

BACKGROUND OF THE INVENTION

Mal de Rio Cuarto Virus (MRCV) is considered to be the most prevalentand destructive viral disease of maize, Zea mays L., in Argentina. MRCVinfection causes abnormal corn development and significantly reducescrop yield. The susceptible phenotype includes stunting, shortenedinternodes, cut and reduced leaves, deformed tassels with no anthers,reduced roots, underdeveloped ears with poor kernel sets and overallthickening of vascular tissues. The largest known outbreak of MRCV inArgentina to date occurred during the 1996/1997 growing season andaffected nearly 300,000 hectares producing approximately $120MM in yieldlosses. MRCV disease is vectored by the leafhopper Delphacodes kuscheli.Increased populations of D. kuscheli in 2006 apparently led to areoccurrence of the viral disease in Argentinean corn plants, whichsignificantly affected the 2007 harvest. Exploratory methods to controlthe disease using pesticides and other means of insect control have beenunsuccessful and development of MCRV tolerant lines through selectivebreeding is a primary initiative for seed producers.

As Bacillus thuringiensis (Bt) technology becomes more widespread inBrazil and Northern Argentina, the amount of insecticide used on corncrops will most likely decrease. This reduction in insecticide mayincrease the numbers of leaf hoppers in the environment, thus amplifyingMRCV disease pressure. Breeding resistance into corn is the principaland most effective control method to manage yield loss associated withMRCV disease. The development of molecular genetic markers hasfacilitated mapping and selection of agriculturally important traits inmaize, and quantitative trait loci (QTL) for MRCV resistance have beenidentified. QTL conferring resistance to MRCV have been identified onchromosomes 1 and 8 (DiRenzo et al. 2004; Kreff et al. 2006), chromosome2 (WO 2009/058335), and chromosomes 4 and 10 (Kreff et al. 2006).Introgression of QTL through the use of molecular markers associatedwith MRCV will increase the speed and accuracy of moving MRCV resistanceinto elite corn hybrids, thus improving the level of resistance insubtropical germplasm. Incorporating MRCV resistance into elite corngermplasm may prevent the spread of the viral disease to non-endemicregions.

Despite the fact that information for MRCV resistance QTL is availablein the art, few pedigrees can be classified as highly tolerant and thereis little evidence of any strong resistance to MRCV in commerciallyavailable hybrids. There is a need for commercially acceptable hybridsthat are MRCV resistant and for a method to develop and track resistantmaize inbreds and hybrids through marker assisted breeding.

Described within is a method to map MRCV resistance QTL in a DHpopulation using a bi-parental QTL mapping approach. The presentinvention allows selection of progeny which contain the genomicbackground of the agronomically desirable parent and the genomic traitof the MRCV resistant donor parent. The present invention also allowstracking of MRCV resistance QTL in order to introgress the MRCVresistance trait into new plants through traditional breeding.

SUMMARY OF THE INVENTION

One embodiment of the present invention is a method for selecting aplant having increased MRCV resistance. The method includes the stepsof: a) detecting at least one marker nucleic acid; and, b) selecting aplant comprising the marker nucleic acid, thereby selecting a planthaving increased MRCV resistance. The plant is preferably a maize plant.

In embodiments of the invention, the marker nucleic acid is selectedfrom the group consisting of PZA02272-3, DAS-PZ-11980, DAS-PZ-8644,DAS-PZ-10816, DAS-PZ-2849, zfl2-9, DAS-PZ-19494, Mo17-11696, andMAGI_105144. In further embodiments of the invention, at least onemarker nucleic acid is selected, and preferably, at least two markernucleic acids are selected.

In another embodiment of the invention is a method for selecting a maizeplant having increased MRCV resistance, the method comprising: a)detecting at least one marker nucleic acids, wherein at least one markernucleic acid is selected from the group consisting of PZA02272-3,DAS-PZ-11980, DAS-PZ-8644, DAS-PZ-10816, DAS-PZ-2849, zfl2-9,DAS-PZ-19494, Mo17-11696, and MAGI_105144; and, b) selecting a plantcomprising at least one marker nucleic acids, thereby selecting a maizeplant having increased MRCV resistance. Maize plants obtained by themethods described herein are also contemplated by the present invention.

BRIEF DESCRIPTION OF FIGURES AND SEQUENCE LISTINGS

The invention can be more fully understood from the following detaileddescription and the accompanying drawings and Sequence Listing, whichform a part of this application. The Sequence Listing contains the oneletter code for nucleotide sequence characters and the three lettercodes for amino acids as defined in conformity with the IUPAC-IUBMBstandards described in Nucleic Acids Research 13:3021-3030 (1985) and inthe Biochemical Journal 219 (No. 2): 345-373 (1984), which are hereinincorporated by reference in their entirety. The symbols and format usedfor nucleotide and amino acid sequence data comply with the rules setforth in 37 C.F.R. §1.822.

SEQ ID NO: 1 contains the PZA02272-3 SNP and flanking sequence.

SEQ ID NO: 2 contains the DAS-PZ-11980 SNP and flanking sequence.

SEQ ID NO: 3 contains the DAS-PZ-8644 SNP and flanking sequence.

SEQ ID NO: 4 contains the DAS-PZ-10816 SNP and flanking sequence.

SEQ ID NO: 5 contains the DAS-PZ-2849 SNP and flanking sequence.

SEQ ID NO: 6 contains the zfl2-9 SNP and flanking sequence.

SEQ ID NO: 7 contains the DAS-PZ-19494 SNP and flanking sequence.

SEQ ID NO: 8 contains the Mo17-11696 SNP and flanking sequence.

SEQ ID NO: 9 contains the MAGI_105144 SNP and flanking sequence.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods for identifying and selectingmaize plants with increased MRCV resistance. The following definitionsare provided as an aid to understand the invention.

The term “allele” refers to one of two or more different nucleotidesequences that occur at a specific locus.

An “amplicon” is amplified nucleic acid, e.g., a nucleic acid that isproduced by amplifying a template nucleic acid by any availableamplification method (e.g., PCR, LCR, transcription, or the like).

The term “amplifying” in the context of nucleic acid amplification isany process whereby additional copies of a selected nucleic acid for atranscribed form thereof are produced. Typical amplification methodsinclude various polymerase based replication methods, including thepolymerase chain reaction (PCR), ligase mediated methods such as theligase chain reaction (LCR) and RNA polymerase based amplification(e.g., by transcription) methods.

The term “assemble” applies to bacterial artificial clones (BACs) andtheir propensities for coming together to form contiguous stretches ofDNA. A BAC “assembles” to a contig based on sequence alignment, if theBAC is sequenced, or via the alignment of its BAC fingerprint to thefingerprints of other BACs. The assemblies can be found using the MaizeGenome Browser, which is publicly available on the internet.

An allele is “associated with” a trait when it is linked to it and whenthe presence of the allele is an indicator that the desired trait ortrait form will occur in a plant comprising the allele.

The “B73 reference genome, version 2” is the physical and geneticframework of the maize B73 genome. It is the result of a sequencingeffort utilizing a minimal tiling path of approximately 19,000 mappedBAC clones, and focusing on producing high-quality sequence coverage ofall identifiable gene-containing regions of the maize genome. Theseregions were ordered, oriented, and along with all of the intergenicsequences, anchored to the extant physical and genetic maps of the maizegenome. It can be accessed using a genome browser, the Maize GenomeBrowser, which is publicly available on the internet and facilitatesuser interaction with sequence and map data.

A “bacterial artificial chromosome (BAC)”is a cloning vector derivedfrom the naturally occurring F factor of Escherichia coli. BACs canaccept large inserts of DNA sequence. In maize, a number of BACs, orbacterial artificial chromosomes, each containing a large insert ofmaize genomic DNA, have been assembled into contigs (overlappingcontiguous genetic fragments, or “contiguous DNA”).

“Backcrossing” refers to the process whereby hybrid progeny arerepeatedly crossed back to one of the parents. In a backcrossing scheme,the “donor” parent refers to the parental plant with the desired gene orlocus to be introgressed. The “recipient” parent (used one or moretimes) or “recurrent” parent (used two or more times) refers to theparental plant into which the gene or locus is being introgressed. Forexample, see Ragot, M. et al. (1995) Marker-assisted backcrossing: apractical example, in Techniques et Utilisations des MarqueursMoleculaires Les Colloques, Vol. 72, pp. 45-56, and Openshaw et al.,(1994) Marker-assisted Selection in Backcross Breeding, Analysis ofMolecular Marker Data, pp. 41-43. The initial cross gives rise to the F1generation: the term “BC1” then refers to the second use of therecurrent parent, “BC2” refers to the third use of the recurrent parent,and so on.

A centimorgan (“cM”) is a unit of measure of recombination frequency.One cM is equal to a 1% chance that a marker at one genetic locus willbe separated from a marker at a second locus due to crossing over in asingle generation.

“Chromosomal interval” designates a contiguous linear span of genomicDNA that resides in plants on a single chromosome. The genetic elementsor genes located on a single chromosomal interval are physically linked.The size of a chromosomal interval is not particularly limited. In someaspects, the genetic elements located within a single chromosomalinterval are genetically linked, typically with a genetic recombinationdistance of, for example, less than or equal to 20 cM, or alternatively,less than or equal to 10 cM. That is, two genetic elements within asingle chromosomal interval undergo recombination at a frequency of lessthan or equal to 20% or 10%, respectively.

The term “chromosomal interval” designates any and all intervals definedby any of the markers set forth in this invention. A chromosomalinterval that correlates with increased MRCV resistance is provided.This interval, located on chromosome 2, comprises and is flanked byPZA02272-3 and MAGI_105144. A subinterval of chromosomal intervalPZA02272-3 and MAGI_105144 is DAS-PZ-2849 and DAS-PZ-19494.

The term “complement” refers to a nucleotide sequence that iscomplementary to a given nucleotide sequence, i.e., the sequences arerelated by the base-pairing rules.

The term “contiguous DNA” refers to overlapping contiguous geneticfragments.

The term “crossed” or “cross” means the fusion of gametes viapollination to produce progeny (e.g., cells, seeds or plants). The termencompasses both sexual crosses (the pollination of one plant byanother) and selfing (self-pollination, e.g., when the pollen and ovuleare from the same plant). The term “crossing” refers to the act offusing gametes via pollination to produce progeny.

The term “elite line” refers to any line that has resulted from breedingand selection for superior agronomic performance. An elite plant is anyplant from an elite line.

A “favorable allele” is the allele at a particular locus that confers,or contributes to, a desirable phenotype, e.g., increased MRCVresistance, or alternatively, is an allele that allows theidentification of plants with decreased MRCV resistance that can beremoved from a breeding program or planting (“counter-selection”). Afavorable allele of a marker is a marker allele that segregates with thefavorable phenotype, or alternatively, segregates with the unfavorableplant phenotype, therefore providing the benefit of identifying plants.

“Fragment” is intended to mean a portion of a nucleotide sequence.Fragments can be used as hybridization probes or PCR primers usingmethods disclosed herein.

A “genetic map” is a description of genetic linkage relationships amongloci on one or more chromosomes (or chromosomes) within a given species,generally depicted in a diagrammatic or tabular form. For each geneticmap, distances between loci are measured by the recombinationfrequencies between them, and recombinations between loci can bedetected using a variety of molecular genetic markers (also calledmolecular markers). A genetic map is a product of the mappingpopulation, types of markers used, and the polymorphic potential of eachmarker between different populations. The order and genetic distancesbetween loci can differ from one genetic map to another. However,information such as marker position and order can be correlated betweenmaps by determining the physical location of the markers on thechromosome of interest, using the B73 reference genome, version 2, whichis publicly available on the internet. One of ordinary skill in the artcan use the publicly available genome browser to determine the physicallocation of markers on a chromosome.

The term “Genetic Marker” shall refer to any type of nucleic acid basedmarker, including but not limited to, Restriction Fragment LengthPolymorphism (RFLP) (Botstein et al, 1998), Simple Sequence Repeat (SSR)(Jacob et al., 1991), Random Amplified Polymorphic DNA (RAPD) (Welsh etal., 1990), Cleaved Amplified Polymorphic Sequences (CAPS) (Rafalski andTingey, 1993, Trends in Genetics 9:275-280), Amplified Fragment LengthPolymorphism (AFLP) (Vos et al, 1995, Nucleic Acids Res. 23:4407-4414),Single Nucleotide Polymorphism (SNP) (Brookes, 1999, Gene 234:177-186),Sequence Characterized Amplified Region (SCAR) (Pecan and Michelmore,1993, Theor. Appl. Genet, 85:985-993), Sequence Tagged Site (STS)(Onozaki et al. 2004, Euphytica 138:255-262), Single StrandedConformation Polymorphism (SSCP) (Orita et al., 1989, Proc Natl Aced SciUSA 86:2766-2770). Inter-Simple Sequence Repeat (ISR) (Blair et al.1999, Theor. Appl. Genet. 98:780-792), Inter-Retrotransposon AmplifiedPolymorphism (TRAP), Retrotransposon-Microsatellite AmplifiedPolymorphism (REMAP) (Kalendar et al., 1999, Theor. Appl. Genet98:704-711), an RNA cleavage product (such as a Lynx tag), and the like.

“Genetic recombination frequency” is the frequency of a crossing overevent (recombination) between two genetic loci. Recombination frequencycan be observed by following the segregation of markers and/or traitsfollowing meiosis.

“Genome” refers to the total DNA, or the entire set of genes, carried bya chromosome or chromosome set.

The term “genotype” is the genetic constitution of an individual (orgroup of individuals) at one or more genetic loci, as contrasted withthe observable trait (the phenotype). Genotype is defined by theallele(s) of one or more known loci that the individual has inheritedfrom its parents. The term genotype can be used to refer to anindividual's genetic constitution at a single locus, at multiple led,or, more generally, the term genotype can be used to refer to anindividual's genetic make-up for all the genes in its genome.

“Germplasm” refers to genetic material of or from an individual (e.g., aplant), a group of individuals (e.g., a plant line, variety or family),or a clone derived from a line, variety, species, or culture. Thegermplasm can be part of an organism or cell, or can be separate fromthe organism or cell. In general, germplasm provides genetic materialwith a specific molecular makeup that provides a physical foundation forsome or all of the hereditary qualities of an organism or cell culture.As used herein, germplasm includes cells, seed or tissues from which newplants may be grown, or plant parts, such as leafs, stems, pollen, orcells that can be cultured into a whole plant.

A “haplotype” is the genotype of an individual at a plurality of geneticloci, i.e. a combination of alleles. Typically, the genetic locidescribed by a haplotype are physically and genetically linked, i.e., onthe same chromosome segment. The term “haplotype” can refer to sequence,polymorphisms at a particular locus, such as a single marker locus, orsequence polymorphisms at multiple loci along a chromosomal segment in agiven genome. The former can also be referred to as “marker haplotypes”or “marker alleles”, while the latter can be referred to as “long-rangehaplotypes”.

The “heritability (h²)” of a trait within a population is the proportionof observable differences in a trait between individuals within apopulation that is due to genetic differences. The h² value of the QTLis a percentage of variation that is explained by genetics, instead ofenvironment.

A “heterotic group” comprises a set of genotypes that perform well whencrossed with genotypes from a different heterotic group (Hallauer at al.(1998) Corn breeding, p. 463-564. In G. F. Sprague and J. W. Dudley (ed)Corn and corn improvement). Inbred lines are classified into heteroticgroups, and are further subdivided into families within a heteroticgroup, based on several criteria such as pedigree, molecularmarker-based associations, and performance in hybrid combinations (Smithat al. (1990) Theor. Appl. Gen. 80:833-840). The two most widely usedheterotic groups in the United States are referred to as “Iowa StiffStalk Synthetic” (BSSS) and “Lancaster” or “Lancaster Sure Crop”(sometimes referred to as NSS, or Iron-Stiff Stalk).

The term “heterozygous” means a genetic condition wherein differentalleles reside at corresponding loci on homologous chromosomes.

The term “homozygous” means a genetic condition wherein identicalalleles reside at corresponding loci on homologous chromosomes.

“Hybridization” or “nucleic acid hybridization” refers to the pairing ofcomplementary RNA and DNA strands as well as the pairing ofcomplementary DNA single strands.

The term “hybridize” means the formation of base pairs betweencomplementary regions of nucleic acid strands.

The term “indel” refers to an insertion or deletion, wherein one linemay be referred to as having an insertion relative to a second line, orthe second line may be referred to as having a deletion relative to thefirst line.

The term “introgression” or “introgressing” refers to the transmissionof a desired allele of a genetic locus from one genetic background toanother. For example, introgression of a desired allele at a specifiedlocus can be transmitted to at least one progeny via a sexual crossbetween two parents of the same species, where at least one of theparents has the desired allele in its genome. Alternatively, forexample, transmission of an allele can occur by recombination betweentwo donor genomes, e.g., in a fused protoplast, where at least one ofthe donor protoplasts has the desired allele in its genome. The desiredallele can be, e.g., a selected allele of a marker, a QTL, a transgene,or the like. In any case, offspring comprising the desired allele can berepeatedly backcrossed to a line having a desired genetic background andselected for the desired allele, to result in the allele becoming fixedin a selected genetic background. For example, the chromosome 2 locusdescribed herein may be introgressed into a recurrent parent that issusceptible to MRCV. The recurrent parent line with the introgressedgene or locus then has increased MRCV resistance.

As used herein, the term “linkage” is used to describe the degree withwhich one marker locus is associated with another marker locus or someother locus (for example, a MRCV locus). The linkage relationshipbetween a molecular marker and a phenotype is given as a “probability”or “adjusted probability”. Linkage can be expressed as a desired limitor range. For example, in some embodiments, any marker is linked(genetically and physically) to any other marker when the markers areseparated by less than 50, 40, 30, 25, 20, or 15 map units for cM). Insome aspects, it is advantageous to define a bracketed range of linkage,for example, between 10 and 20 cM, between 10 and 30 cM, or between 10and 40 cM. The more closely a marker is linked to a second locus, thebetter an indicator for the second locus that marker becomes. Thus,“closely linked loci” such as a marker locus and a second locus displayan inter-locus recombination frequency of 10% or less, preferably about9% or less, still more preferably about 8% or less, yet more preferablyabout 7% or less, still more preferably about 6% or less, yet morepreferably about 5% or less, still more preferably about 4% or less, yetmore preferably about 3% or less, and still more preferably about 2% orless. In highly preferred embodiments, the relevant loci display arecombination frequency of about 1% or less, e.g., about 0.75% or less,more preferably about 0.5% or less, or yet more preferably about 0.25%or less. Two loci that are localized to the same chromosome, and at sucha distance that recombination between the two loci occurs at a frequencyof less than 10 (e.g., about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.75%,0.5%, 0.25%, or less) are also said to be “proximal to” each other.Since one cM is the distance between two markers that show a 1%recombination frequency, any marker is closely linked (genetically andphysically) to any other marker that is in close proximity, e.g., at orless than 10 cM distant. Two closely linked markers on the samechromosome can be positioned 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.75, 0.5 or0.25 cM or less from each other.

The term “linkage disequilibrium” refers to a non-random segregation ofgenetic loci or traits for bothloci. In either case, linkagedisequilibrium implies that the relevant loci are within sufficientphysical proximity along a length of a chromosome so that they segregatetogether with greater than random (i.e., non-random) frequency (in thecase of co-segregating traits, the loci that underlie the traits are insufficient proximity to each other). Markers that show linkagedisequilibrium are considered linked. Linked loci co-segregate more than50% of the time, e.g., from about 51% to about 100% of the time. Inother words, two markers that co-segregate have a recombinationfrequency of less than 50% (and by definition, are separated by lessthan 50 cM on the same chromosome.) As used herein, linkage can bebetween two markers, or alternatively between a marker and a phenotype.A marker locus can be “associated with” (linked to) a trait, e.g.,increased MRCV resistance. The degree of linkage of a molecular markerto a phenotypic trait is measured, e.g. as a statistical probability ofco-segregation of that molecular marker with the phenotype.

Linkage disequilibrium is most commonly assessed using the measure r²,which is calculated using the formula described by Hill, W. G. andRobertson, A, Theor Appl. Genet 38:226-231 (1988). When r²=1, completeLD exists between the two marker loci, meaning that the markers have notbeen separated by recombination and have the same allele frequency.Values for r² above 1/3 indicate sufficiently strong LD to be useful formapping (Ardlie at al., Nature Reviews Genetics 3:299-309 (2002)).Hence, alleles are in linkage disequilibrium when r² values betweenpairwise marker loci are greater than or equal to 0.33, 0.4, 0.5, 0.6,0.7, 0.8, 0.9, or 1.0.

As used herein, “linkage equilibrium” describes a situation where twomarkers independently segregate, i.e., sort among progeny randomly.Markers that show linkage equilibrium are considered unlinked (whetheror not they lie on the same chromosome).

The “logarithm of odds (LOD) value” or “LOD score” (Risch, Science255:803-804 (1992)) is used in interval mapping to describe the degreeof linkage between two marker loci. A LOD score of three between twomarkers indicates that linkage is 1000 times more likely than nolinkage, while a LOD score of two indicates that linkage is 100 timesmore likely than no linkage. LOD scores greater than or equal to two maybe used to detect linkage.

A “locus” is a position on a chromosome where a gene or marker islocated.

“Maize” refers to a plant of the Zea mays L. ssp. mays and is also knownas “corn”.

The term “maize plant” includes: whole maize plants, maize plant cells,maize plant protoplast, maize plant cell or maize tissue cultures fromwhich maize plants can be regenerated, maize plant calli, and maizeplant cells that are intact in maize plants or parts of maize plants,such as maize seeds, maize cobs, maize flowers, maize cotyledons, maizeleaves, maize stems, maize buds, maize roots, maize root tips, and thelike.

“Mal de Rio Cuarto Virus (MRCV)” is a species of virus in the Reoviridaefamily, genus Fijivirus, which causes devastating crop losses forproducers in Argentina.

A “marker” is a nucleotide sequence or encoded product thereof (e.g., aprotein) used as a point of reference. For markers to be useful atdetecting recombinations, they need to detect differences, orpolymorphisms, within the population being monitored. For molecularmarkers, this means differences at the DNA level due to polynucleotidesequence differences (e.g. SSRs, RFLPs, AFLPs, SNPs). The genomicvariability can be of any origin, for example, insertions, deletions,duplications, repetitive elements, point mutations, recombinationevents, or the presence and sequence of transposable elements. Molecularmarkers can be derived from genomic or expressed nucleic acids (e.g.,ESTs) and can also refer to nucleic acids used as probes or primer pairscapable of amplifying sequence fragments via the use of PCR-basedmethods. A large number of maize molecular markers are known in the art,and are published or available from various sources, such as the MaizeGDB Internet resource and the Arizona Genomics Institute Internetresource run by the University of Arizona.

Markers corresponding to genetic polymorphisms between members of apopulation can be detected by methods well-established in the art. Theseinclude, e.g., DNA sequencing, PCR-based sequence specific amplificationmethods, detection of RFLPs, detection of isozyme markers, detection ofpolynucleotide polymorphisms by allele specific hybridization (ASH),detection of amplified variable sequences of the plant genome, detectionof self-sustained sequence replication, detection of SSRs, detection ofSNPs, or detection of AFLPs. Well established methods are also known forthe detection of expressed sequence tags (ESTs) and SSR markers derivedfrom EST sequences and RAPDs.

A “marker allele”, alternatively an “allele of a marker locus”, canrefer to one of a plurality of polymorphic nucleotide sequences found ata marker locus in a population that is polymorphic for the marker locus.

“Marker assisted selection” (MAS) is a process by which phenotypes areselected based on marker genotypes.

“Marker assisted counter-selection” is a process by which markergenotypes are used to identify plants that will not be selected,allowing them to be removed from a breeding program or planting.

A “marker locus” is a specific chromosome location in the genome of aspecies when a specific marker can be found. A marker locus can be usedto track the presence of a second linked locus, e.g., a linked locusthat encodes or contributes to expression of a phenotypic trait. Forexample, a marker locus can be used to monitor segregation of alleles ata locus, such as a QTL or single gene, that are genetically orphysically linked to the marker locus.

A “marker probe” is a nucleic add sequence or molecule that can be usedto identify the presence of a marker locus, e.g., a nucleic acid probethat is complementary to a marker locus sequence, through nucleic addhybridization. Marker probes comprising 30 or more contiguousnucleotides of the marker locus (“all or a portion” of the marker locussequence) may be used for nucleic acid hybridization. Alternatively, insome aspects, a marker probe refers to a probe of any type that is ableto distinguish (i.e. genotype) the particular allele that is present ata marker locus.

The term “molecular marker” may be used to refer to a genetic marker, asdefined above, or an encoded product thereof (e.g., a protein) used as apoint of reference when identifying a linked locus. A marker can bederived from genomic nucleotide sequences or from expressed nucleotidesequences (e.g., from a spliced RNA, a cDNA, etc.), or from an encodedpolypeptide. The term also refers to nucleic acid sequencescomplementary to or flanking the marker sequences, such as nucleic acidsused as probes or primer pairs capable of amplifying the markersequence. A “molecular marker probe” is a nucleic acid sequence ormolecule that can be used to identify the presence of a marker locus,e.g., a nucleic acid probe that is complementary to a marker locussequence. Alternatively, in some aspects, a marker probe refers to aprobe of any type that is able to distinguish (i.e., genotype) theparticular allele that is present at a marker locus. Nucleic acids are“complementary” when they specifically hybridize in solution, e.g.,according to Watson-Crick base pairing rules. Some of the markersdescribed herein are also referred to as hybridization markers whenlocated on an indel region, such as the non-collinear region describedherein. This is because the insertion region is, by definition, apolymorphism vis a via a plant without the insertion. Thus, the markerneed only indicate whether the indel region is present or absent. Anysuitable marker detection technology may be used to identify such ahybridization marker, e.g., SNP technology is used in the examplesprovided herein.

The “Non-Stiff Stalk” heterotic group represents a major temperateheterotic group. It can also be referred to as the non-Iowa Stiff StalkSynthetic for BSSS (non-BSSS) heterotic group.

“Nucleotide sequence”, “polynucleotide”, “nucleic acid sequence”, and“nucleic acid fragment” are used interchangeably and refer to a polymerof RNA or DNA that is single- or double-stranded, optionally containingsynthetic, non-natural or altered nucleotide bases. A “nucleotide” is amonomeric unit from which DNA or RNA polymers are constructed, andconsists of a purine or pyrimidine base, a pentose, and a phosphoricacid group. Nucleotides (usually found in their 5′-monophosphate form)are referred to by their single letter designation as follows: “A” foradenylate or deoxyadenylate (for RNA or DNA, respectively), “C” forcytidylate or deoxycytidylate. “G” for guanylate or deoxyguanylate. “U”for uridylate, “T” for deoxythymidylate, “R” for purines (A or G), “Y”for pyrimidines (C or T), “K” for G or T, “H” for A or C or T, “I” forinosine, and “N” for any nucleotide.

The terms “phenotype”, or “phenotypic trait” or “trait” refers to one ormore traits of an organism. The phenotype can be observable to the nakedeye, or by any other means of evaluation known in the art, e.g.,microscopy, biochemical analysis, or an electromechanical assay. In somecases, a phenotype is directly controlled by a single gene or geneticlocus, i.e., a “single gene trait”. In other cases, a phenotype is theresult of several genes.

A “physical map” of the genome is a map showing the linear order ofidentifiable landmarks (including genes, markers, etc.) on chromosomeDNA. However, in contrast to genetic maps, the distances betweenlandmarks are absolute (for example, measured in base pairs or isolatedand overlapping contiguous genetic fragments) and not based on geneticrecombination.

A “plant” can be a whole plant, any part thereof, or a cell or tissueculture derived from a plant. Thus, the term “plant” can refer to anyof: whole plants, plant components or organs (e.g., leaves, stems,roots, etc.), plant tissues, seeds, plant cells, and/or progeny of thesame. A plant cell is a cell of a plant, taken from a plant, or derivedthrough culture from a cell taken from a plant.

A “polymorphism” is a variation in the DNA that is too common to be duemerely to new mutation. A polymorphism must have a frequency of at least1% in a population. A polymorphism can be a single nucleotidepolymorphism, or SNP, or an insertion/deletion polymorphism, alsoreferred to herein as an “indel”.

The “probability value” or “p-value” is the statistical likelihood thatthe particular combination of a phenotype and the presence or absence ofa particular marker allele is random. Thus, the lower the probabilityscore, the greater the likelihood that a phenotype and a particularmarker will co-segregate. In some aspects, the probability score isconsidered “significant” or “nonsignificant”. In some embodiments, aprobability score of 0.05 (p=0.05, or a 5% probability) of randomassortment is considered a significant indication of co-segregation.However, an acceptable probability can be any probability of less than50% (p=0.5). For example, a significant probability can be less than0.25, less than 0.20, less than 0.15, less than 0.1, less than 0.05,less than 0.01, or less than 0.001.

The term “progeny” refers to the offspring generated from a cross.

A “progeny plant” is generated from a cross between two plants.

A “reference sequence” is a defined sequence used as a basis forsequence comparison. The reference sequence is obtained by genotyping anumber of lines at the locus, aligning the nucleotide sequences in asequence alignment program (e.g. Sequencher), and then obtaining theconsensus sequence of the alignment.

A “single nucleotide polymorphism (SNP)” is an allelic singlenucleotide-A, T, C or G-variation within a DNA sequence representing onelocus of at least two individuals of the same species,. For example, twosequenced DNA fragments representing the same locus from at least twoindividuals of the same species, AAGCCTA to AAGCTTA, contain adifference in a single nucleotide.

The phrase “under stringent conditions” refers to conditions under whicha probe or polynucleotide will hybridize to a specific nucleic acidsequence, typically in a complex mixture of nucleic acids, but toessentially no other sequences. Stringent conditions aresequence-dependent and will be different in different circumstances.

Sequence alignments and percent identity calculations may be determinedusing a variety of comparison methods designed to detect homologoussequences including, but not limited to, the MEGALIGN® program of theLASERGENE® bioinformatics computing suite (DNASTAR® Inc., Madison,Wis.). Unless stated otherwise, multiple alignment of the sequencesprovided herein were performed using the Clustal V method of alignment(Higgins and Sharp, CABIOS. 5:151-153 (1989)) with the defaultparameters (GAP PENALTY=10, GAP LENGTH PENALTY=10), Default parametersfor pairwise alignments and calculation of percent identity of proteinsequences using the Clustal V method are KTUPLE=1, GAP PENALTY=3,WINDOW=5 and DIAGONALS SAVED=5. For nucleic adds these parameters areKTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4. After alignmentof the sequences, using the Clustal V program, it is possible to obtain“percent identity” and “divergence” values by viewing the “sequencedistances” table on the same program; unless stated otherwise, percentidentities and divergences provided and claimed herein were calculatedin this manner.

Before describing the present invention in detail, it should beunderstood that this invention is not limited to particular embodiments.It also should be understood that the terminology used herein is for thepurpose of describing particular embodiments, and is not intended to belimiting. As used herein and in the appended claims, terms in thesingular and the singular forms “a”, “an” and “the”, for example,include plural referents unless the content clearly dictates otherwise.Thus, for example, reference to “plant”, “the plant” or “a plant” alsoincludes a plurality of plants. Depending on the context, use of theterm “plant” can also include genetically similar or identical progenyof that plant. The use of the term “a nucleic acid” optionally includesmany copies of that nucleic acid molecule.

Genetic Mapping

It has been recognized for quite some time that specific genetic locicorrelating with particular phenotypes, such as increased MRCVresistance, can be mapped in an organism's genome. The plant breeder canadvantageously use molecular markers to identify desired individuals bydetecting marker alleles that show a statistically significantprobability of co-segregation with a desired phenotype, manifested aslinkage disequilibrium. By identifying a molecular marker or clusters ofmolecular markers that co-segregate with a trait of interest, thebreeder is able to rapidly select a desired phenotype by selecting forthe proper molecular marker allele (a process called marker-assistedselection, or MAS).

A variety of methods well known in the art are available for detectingmolecular markers or clusters of molecular markers that co-segregatewith a trait of interest, such as increased MRCV resistance. The basicidea underlying these methods is the detection of markers, for whichalternative genotypes (or alleles) have significantly different averagephenotypes. Thus, one makes a comparison among marker loci of themagnitude of difference among alternative genotypes (or alleles) or thelevel of significance of that difference. Trait genes are inferred to belocated nearest the marker(s) that have the greatest associatedgenotypic difference.

Two such methods used to detect trait loci of interest are: 1)Population-based association analysis and 2) Traditional linkageanalysis. In a population-based association analysis, lines are obtainedfrom pre-existing populations with multiple founders, e.g. elitebreeding lines. Population-based association analyses rely on the decayof linkage disequilibrium (LD) and the idea that in an unstructuredpopulation, only correlations between genes controlling a trait ofinterest and markers closely linked to those genes will remain after somany generations of random mating. In reality, most pre-existingpopulations have population substructure. Thus, the use of a structuredassociation approach helps to control population structure by allocatingindividuals to populations using data obtained from markers randomlydistributed across the genome, thereby minimizing disequilibrium due topopulation structure within the individual populations (also calledsubpopulations). The phenotypic values are compared to the genotypes(alleles) at each, marker locus for each line in the subpopulation. Asignificant marker-trait association indicates the dose proximitybetween the marker locus and one or more genetic loci that are involvedin the expression of that trait.

The same principles underlie traditional linkage analysis; however, LDis generated by creating a population from a small number of founders.The founders are selected to maximize the level of polymorphism withinthe constructed population, and polymorphic sites are assessed for theirlevel of cosegregation with a given phenotype. A number of statisticalmethods have been used to identify significant marker-traitassociations. One such method is an interval mapping approach (Landerand Botstein, Genetics 121:185-199 (1989), in which each of manypositions along a genetic map (say at 1 cM intervals) is tested for thelikelihood that a gene controlling a trait of interest is located atthat position. The genotype/phenotype data are used to calculate foreach test position a LOD score (log of likelihood ratio). When the LODscore exceeds a threshold value, there is significant evidence for thelocation of a gene controlling the trait of interest at that position onthe genetic map (which will fall between two particular marker loci).

Markers Associated with MRCV

Markers associated with increased MRCV resistance are identified herein.The methods involve detecting the presence of at least one marker alleleassociated with the enhanced resistance in the germplasm of a maizeplant. The marker locus can be selected from any of the marker lociprovided in Table 3, including PZA02272-3 and MAGI_105144, and any othermarker linked to these markers (linked markers can be determined fromthe Maize GDB resource). The marker locus can be selected from any ofthe marker loci provided in Table 3, including PZA02272-3, DAS-PZ-11980,DAS-PZ-8644, DAS-PZ-10816, DAS-PZ-2849, zfl2-9, DAS-PZ-19494,Mo17-11696, and MAGI_105144 and any other marker linked to this marker(linked markers can be determined from the Maize GDB resource).

The genetic elements or genes located on a contiguous linear span ofgenomic DNA on a single chromosome are physically linked. PZA02272-3 andMAGI_105144, both highly associated with MRCV resistance, delineate anMRCV resistance QTL. Any polynucleotide that assembles to the contiguousDNA between and including SEQ ID NO:1 (the reference sequence forPZA02272-3), or a nucleotide sequence that is 95% identical to SEQ IDNO: 1 based on the Clustal V method of alignment, and SEQ ID NO:9 (thereference sequence for MAGI_105144), or a nucleotide sequence that is95% identical to SEQ ID NO:9 based on the Clustal V method of alignment,can house marker loci that are associated with MRCV resistance.

The genetic elements or genes located on a contiguous linear span ofgenomic DNA on a single chromosome are physically linked for thesubinterval of DAS-PZ-2849 and DAS-PZ-19494. DAS-PZ-2849 andDAS-PZ-19494, both highly associated with MRCV resistance, delineate aMRCV resistance QTL. Any polynucleotide that assembles to the contiguousDNA between and including SEQ ID NO:5 (the reference sequence forDAS-PZ-2849), or a nucleotide sequence that is 95% identical to SEQ IDNO:5 based on the Clustal V method of alignment, and SEQ ID NO:7 (thereference sequence for DAS-PZ-19494), or a nucleotide sequence that 95%identical to SEQ ID NO:7 based on the Clustal V method of alignment, canhouse marker loci that are associated with MRCV resistance.

A common measure of linkage is the frequency with which traitscosegregate. This can be expressed as a percentage of cosegregation(recombination frequency) or in centiMorgans (cM). The cM is a unit ofmeasure of genetic recombination frequency. One cM is equal to a 1%chance that a trait at one genetic locus will be separated from a traitat another locus due to crossing over in a single generation (meaningthe traits segregate together 99% of the time). Because chromosomaldistance is approximately proportional to the frequency of crossing overevents between traits, there is an approximate physical distance thatcorrelates with recombination frequency.

Marker loci are themselves traits and can be assessed according tostandard linkage analysis by tracking the marker loci duringsegregation. Thus, one cM is equal to a 1% chance that a marker locuswill be separated from another locus, due to crossing over in a singlegeneration.

Other markers linked to the markers listed in Table 3 can be used topredict MRCV resistance in a maize plant. This includes any markerwithin 50 cM of PZA02272-3, DAS-PZ-11980, DAS-PZ-8644, DAS-PZ-10816,DAS-PZ-2849, zfl2-9, DAS-PZ-19494, Mo17-11696, and MAGI_105144, themarkers associated with MRCV resistance. The closer a marker is to agene controlling a trait of interest, the more effective andadvantageous that marker is as an indicator for the desired trait.Closely linked loci display an inter-locus cross-over frequency of about10% or less, preferably about 9% or less, still more preferably about 8%or less, yet more preferably about 7% or less, still more preferablyabout 6% or less, yet more preferably about 5% or less, still morepreferably about 4% or less, yet more preferably about 3% or less, andstill more preferably about 2% or less. In highly preferred embodiments,the relevant loci (e.g., a marker locus and a target locus) display arecombination frequency of about 1% or less, e.g., about 0.75% or less,more preferably about 0.5% or less, or yet more preferably about 0.25%or less. Thus, the loci are about 10 cM, 9 cM, 8 cM, 7 cM, 6 cM, 5 cM, 4cM, 3 cM, 2 cM, 1 cM, 0.75 cM, 0.5 cM or 0.25 cM or less apart. Putanother way, two loci that are localized to the same chromosome, and atsuch a distance that recombination between the two loci occurs at afrequency of less than 10% (e.g., about 9%, 8% 7%, 6%, 5%, 4%, 3%, 2%1%, 0.75%, 0.5%, 0.25.degree., or less) are said to be “proximal to”each other.

Although particular marker alleles can show co-segregation withincreased MRCV resistance, it is important to note that the marker locusis not necessarily responsible for the expression of the MRCV resistancephenotype. For example, it is not a requirement that the markerpolynucleotide sequence be part of a gene that imparts increased MRCVresistance (for example, be part of the gene open reading frame). Theassociation between a specific marker allele and the increased MRCVresistance phenotype is due to the original “coupling” linkage phasebetween the marker allele and the allele in the ancestral maize linefrom which the allele originated. Eventually, with repeatedrecombination, crossing over events between the marker and genetic locuscan change this orientation. For this reason, the favorable markerallele may change depending on the linkage phase that exists within theresistant parent used to create segregating populations. This does notchange the fact that the marker can be used to monitor segregation ofthe phenotype. It only changes which marker allele is consideredfavorable in a given segregating population.

The term “chromosomal interval” designates any and all intervals definedby any of the markers set forth in this invention. A chromosomalinterval that correlates with MRCV resistance is provided. Thisinterval, located on chromosome 2, comprises and is flanked byPZA02272-3 and MAGI_105144. A subinterval of chromosomal intervalPZA02272-3 and MAGI_105144 is DAS-PZ-2849 and DAS-PZ-19494.

A variety of methods well known in the art are available for identifyingchromosomal intervals. The boundaries of such chromosomal intervals aredrawn to encompass markers that will be linked to the gene controllingthe trait of interest. In other words, the chromosomal interval is drawnsuch that any marker that lies within that interval (including theterminal markers that define the boundaries of the interval) can be usedas a marker for MRCV resistance. The interval described aboveencompasses a cluster of markers that co-segregate with MRCV resistance.The clustering of markers occurs in relatively small domains on thechromosomes, indicating the presence of a gene controlling the trait ofinterest in those chromosome regions.

The interval was drawn to encompass the markers that co-segregate withMRCV resistance. The interval encompasses markers that map within theinterval as well as the markers that define the termini. For example,PZA02272-3 and MAGI_105144, separated by 7834272 bp based on the B73reference genome, version 2, define a chromosomal interval encompassinga cluster of markers that co-segregate with MRCV resistance. A secondexample includes the subinterval, DAS-PZ-2849 and DAS-PZ-19494,separated by 1402949 bp based on the B73 reference genome, version 2,which defines a chromosomal interval encompassing a cluster of markersthat co-segregate with MRCV resistance. An interval described by theterminal markers that define the endpoints of the interval will includethe terminal markers and any marker localizing within that chromosomaldomain, whether those markers are currently known or unknown.

Chromosomal intervals can also be defined by markers that are linked to(show linkage disequilibrium with) a marker of interest, and is a commonmeasure of linkage disequilibrium (LD) in the context of associationstudies. If the r² value of LD between any chromosome 2 marker locuslying within the interval of PZA02272-3 and MAGI_105144, the subintervalof DAS-PZ-2849 and DAS-PZ-19494, or any other subinterval of PZA02272-3and MAGI_105144, and an identified marker within that interval that hasan allele associated with increased MRCV resistance is greater than 1/3(Ardlie et al. Nature Reviews Genetics 3:299-309 (2002)), the loci arelinked.

A marker of the invention can also be a combination of alleles at markerloci, otherwise known as a haplotype. The skilled artisan would expectthat there might be additional polymorphic sites at marker loci in andaround the chromosome 2 markers identified herein, wherein one, or morepolymorphic sites is in linkage disequilibrium (LD) with an alleleassociated with increased MRCV resistance. Two particular alleles atdifferent polymorphic sites are said to be in LD if the presence of theallele at one of the sites tends to predict the presence of the alleleat the other site on the same chromosome (Stevens, Mol. Diag. 4:309-17(1999)).

Marker Assisted Selection

Molecular markers can be used in a variety of, plant breedingapplications (e.g. see Staub et al. (1996) Hortscience 729-741; Tanksley(1983) Plant Molecular Biology Reporter 1: 3-8). One of the main areasof interest is to increase the efficiency of backcrossing andintrogressing genes using marker-assisted selection (MAS). A molecularmarker that demonstrates linkage with a locus affecting a desiredphenotypic trait provides a useful tool for the selection of the traitin a plant population. This is particularly true with traits that aredifficult to phenotype due to their dependence on environmentalconditions. This category includes traits related to the resistance tobiotic and abiotic stresses. This category also includes traits that arevery expensive to phenotype because of laborious artificial inoculationor maintenance of managed stress environments. Another category oftraits includes those which are associated with destruction of plant perse. Destructive phenotyping has been a bottleneck to implement MAS forthe seed quality traits. Because DNA marker assays are notenvironmentally dependent, are robust, reliable, less laborious, lesscostly and take up less physical space than field phenotyping, muchlarger populations can be assayed, increasing the chances of finding arecombinant with the target segment from the donor line moved to therecipient line. The closer the linkage, the more useful the marker, asrecombination is less likely to occur between the marker and the genecausing the trait, which can result in false positives. Having flankingmarkers decreases the chances that false positive selection will occuras a double recombination event would be needed. The ideal situation isto have a marker in the gene itself, so that recombination cannot occurbetween the marker and the gene. Such a marker is called a ‘perfectmarker’.

When a gene is introgressed by MAS, it is not only the gene that isintroduced but also the flanking regions (Gepts. (2002). Crop Sci; 42:1780-1790). This is referred to as “linkage drag.” In the case where thedonor plant is highly unrelated to the recipient plant, these flankingregions carry additional genes that may code for agronomicallyundesirable traits. This “linkage drag” may also result in reduced yieldor other negative agronomic characteristics even after multiple cyclesof backcrossing into the elite maize line. This is also sometimesreferred to as “yield drag.” The size of the flanking region can bedecreased by additional backcrossing, although this is not alwayssuccessful, as breeders do not have control over the size of the regionor the recombination breakpoints (Young et al. (1998) Genetics120:579-585). In classical breeding it is usually only by chance thatrecombinations are selected that contribute to a reduction in the sizeof the donor segment (Tanksley et al. (1989). Biotechnology 7: 257-264).

Even after 20 backcrosses in backcrosses of this type, one may expect tofind a sizeable piece of the donor chromosome still linked to the genebeing selected. With markers however, it is possible to select thoserare individuals that have experienced recombination near the gene ofinterest. In 150 backcross plants, there is a 95% chance that at leastone plant will have experienced a crossover within 1 cM of the gene,based on a single meiosis map distance. Markers will avow unequivocalidentification of those individuals. With one additional backcross of300 plants, there would be a 95% chance of a crossover within 1 cMsingle meiosis map distance of the other side of the gene, generating asegment around the target gene of less than 2 cM based on a singlemeiosis map distance. This can be accomplished in two generations withmarkers, while it would have required on average 100 generations withoutmarkers (See Tanksley et al., supra). When the exact location of a geneis known, flanking markers surrounding the gene can be utilized toselect for recombinations in different population sizes. For example, insmaller population sizes, recombinations may be expected further awayfrom the gene, so more distal flanking markers would be required todetect the recombination.

The availability of the B73 reference genome, version 2 and theintegrated linkage maps of the maize genome containing increasingdensities of public maize markers, has facilitated maize genetic mappingand MAS. See, e.g. the IBM2 Neighbors maps, which are available onlineon the Maize GDB website.

The key components to the implementation of MAS are (i) Defining thepopulation within which the marker-trait association will be determined,which can be a segregating population, or a random or structuredpopulation; (ii) monitoring the segregation or association ofpolymorphic markers relative to the trait, and determining linkage orassociation using statistical methods; (iii) defining a set of desirablemarkers based on the results of the statistical analysis, and (iv) theuse and/or extrapolation of this information to the current set ofbreeding germplasm to enable marker-based selection decisions to bemade. The markers described in this disclosure, as well as other markertypes such as SSRs and FLPs, can be used in marker assisted selectionprotocols.

SSRs can be defined as relatively short runs of tandemly repeated DNAwith lengths of 6 bp or less (Tautz (1989) Nucleic Acid Research 17:6463-6471; Wang et al. (1994) Theoretical and Applied Genetics, 88:1-6).Polymorphisms arise due to variation in the number of repeat units,probably caused by slippage during DNA replication (Levinson and Gutman(1987) Mol Biol Evol 4: 203-221). The variation in repeat length may bedetected by designing PCR primers to the conserved non-repetitiveflanking regions (Weber and May (1989) Am J Hum Genet. 44:388-396), SSRsare highly suited to mapping and MAS as they are multi-allelic,codominant, reproducible and amenable to high throughput automation(Rafalski et al. (1996) Generating and using DNA markers in plants. InNon-mammalian genomic analysis: a practical guide. Academic Press, pp75-135).

Various types of SSR markers can be generated, and SSR profiles fromresistant lines can be obtained by gel electrophoresis of theamplification products. Scoring of marker genotype is based on the sizeof the amplified fragment. An SSR service for maize is available to thepublic on a contractual basis by DNA Landmarks inSaint-Jean-sur-Richelieu, Quebec, Canada.

Various types of FLP markers can also be generated. Most commonly,amplification primers are used to generate fragment lengthpolymorphisms. Such FLP markers are in many ways similar to SSR markers,except that the region amplified by the primers is not typically ahighly repetitive region. Still, the amplified region, or amplicon, willhave sufficient variability among germplasm, often due to insertions ordeletions, such that the fragments generated by the amplificationprimers can be distinguished among polymorphic individuals, and suchindels are known to occur frequently in maize (Bhattramakki et al.(2002). Plant Mol Biol 48, 539-547; Rafalski (2002b), supra).

SNP markers detect single base pair nucleotide substitutions. Of all themolecular marker types, SNPs are the most abundant, thus having thepotential to provide the highest genetic map resolution (Bhattramakki etal. 2002 Plant Molecular Biology 48:539-547). SNPs can be assayed at aneven higher level of throughput than SSRs, in a so-called‘ultra-high-throughput’ fashion, as they do not require large amounts ofDNA and automation of the assay may be straight-forward. SNPs also havethe promise of being relatively low-cost systems. These three factorstogether make SNPs highly attractive for use in MAS. Several methods areavailable for SNP genotyping, including but not limited to,hybridization, primer extension, oligonucleotide ligation, nucleasecleavage, minisequencing and coded spheres. Such methods have beenreviewed in: Gut (2001) Hum Mutat 17 pp, 475-492: Shi (2001) Clin Chem47, pp. 164-172; Kwok (2000) Pharmacogenomics 1, pp. 95-100:Bhattramakki and Rafalski (2001) Discovery and application of singlenucleotide polymorphism markers in plants. In: R, J Henry, Ed, PlantGenotyping: The DNA Fingerprinting of Plants, CABI Publishing,VVallingford. A wide range of commercially available technologiesutilize these and other methods to interrogate SNPs including Masscode™.(Qiagen), Invader® (Third Wave Technologies), SnapShot® (AppliedBiosystems), Taqman® (Applied Biosystems) and Beadarrays™ (Illumina).

A number of SNPs together within a sequence, or across linked sequences,can be used to describe a haplotype for any particular genotype (Chinget al. (2002), BMC Genet. 3:19 pp Gupta et al. 2001, Rafalski (2002b),Plant Science 162:329-333). Haplotypes can be more informative than,single SNPs and can be more descriptive of any particular genotype. Forexample, single SNP may be allele ‘T’ for a specific line or varietywith increased GS tolerance, but the allele might also occur in themaize breeding population being utilized for recurrent parents. In thiscase, a haplotype, e.g. a combination of alleles at linked SNP markers,may be more informative. Once a unique haplotype has been assigned to adonor chromosomal region, that haplotype can be used in that populationor any subset thereof to determine whether an individual has aparticular gene. See, for example, WO2003054229. Using automated highthroughput marker detection platforms known to those of ordinary skillin the art makes this process highly efficient and effective.

The sequences listed in Table 3 can be readily used to obtain additionalpolymorphic SNPs (and other markers) within the QTL interval listed inthis disclosure. Markers within the described map region can behybridized to BACs or other genomic libraries, or electronically alignedwith genome sequences, to find new sequences in the same approximatelocation as the described markers.

In addition to SSRs, FLPs and SNPs, as described above, other types ofmolecular markers are also widely used, including but not limited to,markers derived from EST sequences, RAPDs, and other nucleic acid basedmarkers.

Isozyme profiles and linked morphological characteristics can, in somecases, also be indirectly used as markers. Even though they do notdirectly detect DNA differences, they are often influenced by specificgenetic differences. However, markers that detect DNA variation are farmore numerous and polymorphic than isozyme or morphological markers(Tanksley (1983) Plant Molecular Biology Reporter 1:3-8).

Sequence alignments or contigs may also be used to find sequencesupstream or downstream of the specific markers listed herein. These newsequences, close to the markers described herein, are then used todiscover and develop functionally equivalent markers. For example,different physical and/or genetic maps are aligned to locate equivalentmarkers not described within this disclosure but that are within similarregions. These maps may be within the maize species, or even acrossother species whose genomes share some level of colinearity at macro-and micro-level with maize, such as rice and sorghum.

In general, MAS uses polymorphic markers that have been identified ashaving a significant likelihood of co-segregation with MRCV resistance.Such markers are presumed to map near quantitative trait loci (QTL),give the plant its MRCV resistant phenotype, and are consideredindicators, or markers, for the desired trait. Markers test maize plantsfor the presence of a desired allele, and those which contain a desiredgenotype at one or more loci are expected to transfer the desiredgenotype, along with a desired phenotype, to their progeny. The means toidentify maize plants that have increased MRCV resistance by identifyingplants that have a specified allele at any one of marker loci describedherein, including PZA02272-3, DAS-PZ-11980, DAS-PZ-8644, DAS-PZ-10816,DAS-PZ-2849, zfl2-9, DAS-PZ-19494, Mo17-11696, and MAGI_105144 arepresented herein.

The interval presented herein finds use in MAS to select plants thatdemonstrate increased MRCV resistance. Any marker that maps within thechromosome 2 interval defined by and including PZA02272-3 andMAGI_105144 can be used for this purpose. In addition, haplotypescomprising alleles at one or more marker loci within the chromosome 2interval defined by and including PZA02272-3 and MAGI_105144 can be usedto introduce increased MRCV resistance into maize lines or varieties.Any allele or haplotype that is in linkage disequilibrium with an alleleassociated with increased MRCV resistance can be used in MAS to selectplants with increased MRCV resistance.

EXAMPLES

The following examples are offered to illustrate, but not to limit, theappended claims. It is understood that the examples and embodimentsdescribed herein are for illustrative purposes only and that personsskilled in the art will recognize various reagents or parameters thatcan be altered without departing from the spirit of the invention or thescope of the appended claims.

Example 1 Plant Material

A Dow AgroSciences (DAS) elite line consistently displayed high levelsof Mal de Rio Cuarto Virus (MRCV) resistance under infestationconditions. To identify QTL associated with this line that display ahigh correlation to MRCV resistance, a doubled haploid (DH) mappingpopulation was developed and phenotypically evaluated in two endemicregions known to have high levels of viral outbreaks. QTL mappinganalyses were completed using data collected over two successive yearsfrom the DH populations (Table1).

TABLE 1 Population size and SNP marker counts for the DH populationevaluated for resistance to MRCV disease over two successive years.Mapping SNP Marker Populations Year Sample Size Count DH Population 2010163 509 DH Population 2011 173 446

Example 2 MRCV Disease Phenotype Evaluation

The DH mapping population was evaluated for MRCV symptomology in twoendemic environments, Sampacho and Suco, Argentina. Two replicate samplepopulations were designed as randomized complete block studies (RCB).Plots were arranged in either two 3 meter (m) rows, or one 6 m row, withnearly 25 plants per DH line. In 2010, populations were planted on twoseparate dates to increase the chance of matching the stage of highestsusceptibility of the corn plants with the highest peak of the vectorpopulation. In 2011, all populations were planted on three differentplanting dates spaced approximately one week apart for the same reason.Herbicide and fungicide treatments were used as needed for preventivecontrol. In 2010, disease symptoms were evaluated at 20 days afterflowering. In 2011, MRCV symptomology was assessed at three differenttime points over the course of the season in an effort to capture thetimeframe that most accurately demonstrated a variable range of diseaseincidence. The intention was to capture phenotype data before diseasepressure reached saturation. In both years, disease ratings werecharacterized using a percent incidence and a severity scale (MDG, MeanDisease Grade) as follows:

MDG=Σ _(i=0) ⁵fi.xi

where fi: frequency of grade i, and xi: value of i-th grade. Grades areshown in Table 2.

TABLE 2 Descriptions of the Mean Disease Grade of MRCV. Grade Symptoms 0healthy plants; no symptoms 1 slight symptoms in upper leaves andtassels 2 moderate symptoms, slight height reduction, shortening ofupper internodes and ear 3 stronger height reduction, multiple andconical ears, plant still productive but reduced yield 4 plant heightseverely reduced, multiple ears, scarce or null grain production due toear reduction and malformations 5 dead plants; if still alive, extremedwarfism, absence of ears, upper leaves and tassel

Example 3 DNA Extraction and Single Nucleotide Polymorphic (SNP)Analysis

JoinMap® 3.0 (Van Ooijen et al., 2001) was used to develop a linkage mapfor subsequent QTL analysis. Interval mapping and composite intervalmapping was conducted using MapQTL® 5.0 (Van Ooijen et al., 2002). Apermutation test consisting of 1000 iterations was completed todetermine the significant logarithm-of-odds (LOD) threshold value usinga genome-widep value of 0.05. Loci with LOD scores greater than thecalculated significant threshold were identified as potential QTL. Theposition with the largest LOD value on the linkage group was used as theestimated position of the QTL on the map.

A QTL was identified on chromosome 2 in the DH mapping population forboth the Sampacho and Suco 2010 and 2011 data sets. The QTL was detectedusing the averaged 2011 phenotypic data from Sampacho and explained27.9% of the variation for that environment. The QTL was detected usingthe averaged 2011 data from Suco and explained 45.9% of the variation inthat environment. The chromosome 2 QTL is defined by the interval ofPZA02272-3 and MAGI_105144, with the QTL peak defining the subintervalof DAS-PZ-2849 and DAS-PZ-19494 (Table 3).

Example 4 Marker Framework and Use for MAS

A set of common markers can be used to establish a framework foridentifying markers in the QTL interval. Table 3 shows markers that arein consistent position relative to one another on the DAS internallyderived map and the B73 reference genome, version 2. Physical locationsof the DAS proprietary markers were determined using DAS proprietaryGBrowser.

Closely linked markers flanking the locus of interest that have allelesin linkage disequilibrium with a favorable allele at that locus may beeffectively used to select for progeny plants with increased MRCVresistance. Thus, the markers described herein, such as those listed inTable 3, as well as other markers genetically or physically mapped tothe same chromosomal segment, may be used to select for maize plantswith increased MRCV resistance. Typically, a set of these markers willbe used (e.g. 2 or more, 3 or more, 4 or more, 5 or more) in the regionsflanking the locus of interest. Optionally, a marker within the actualgene and/or locus may be used.

TABLE 3 Chromosome 2 interval and markers associated with MRCV resistance. SEQ Physical ID Donor Position MarkerNO. SNP Allele (bp) PZA02272-3 1 A/G A  9962933 DAS-PZ-11980 2 T/A T11217305 DAS-PZ-8644 3 C/G C 11418854 DAS-PZ-10816 4 G/A G 12103033DAS-PZ-2849 5 A/G A 12255007 zfl2-9 6 A/C C 12644166 DAS-PZ-19494 7 A/TA 13657956 Mo17-11696 8 T/C T 15884180 MAGI_105144 9 A/G G 17797205

Physical positions were determined from the B73 reference genome,version 2.

REFERENCES

Di Renzo, M. A., Bonamico, N. C., Diaz, D. D., Ibanez, M. A., Faricell,M. E., Balzarini M. G. and Salerno, S. J. (2004). Microsatellite markerslinked to QTL for resistance to Mal de Rio Cuarto disease in Zea mays L.J Agric Sci, 142:289-295.

Kreff, E.D., Pacheco, M. G., Diaz, D. G., Robredo, C. G., Puecher, D.,Celiz, A., and Salerno, J. C. (2006). Resistance to Mal de Rio CuartoVirus in maize: A QTL analysis. J Basic Appl Genet 17:41-50.

Martin, T., Franchino, J. A., Kreff, E. D., Procopiuk, A. M., Tomas, A.,Luck, S. D., Shu, G. G. (2009). Major QTLs co

We claim:
 1. A method for producing a plant that displays increased MRCVresistance, the method comprising the steps of: detecting at least onemarker nucleic acid; selecting a plant comprising the marker nucleicacid, thereby selecting a plant that displays increased MRCV resistance;and propagating the selected plant, thereby producing a plant thatdisplays increased MRCV resistance.
 2. The method of claim 1, whereinthe plant is a maize plant.
 3. The method of claim 2, wherein the markernucleic acid is selected from the group consisting of PZA02272-3,DAS-PZ-11980, DAS-PZ-8644, DAS-PZ-10816, DAS-PZ-2849, zfl2-9,DAS-PZ-19494, Mo17-11696, and MAGI_105144.
 4. The method of claim 1,wherein at least two marker nucleic acids are selected.
 5. The method ofclaim 1, wherein at least three marker nucleic acids are selected. 6.The method of claim 1, wherein at least four marker nucleic acids areselected.
 7. A method for producing a maize plant having increased MRCVresistance, the method comprising detecting at least one marker nucleicacid, wherein at least one marker nucleic acid is selected from thegroup consisting of PZA02272-3, DAS-PZ-11980, DAS-PZ-8644, DAS-PZ-10816,DAS-PZ-2849, zfl2-9, DAS-PZ-19494, Mo17-11696, and MAGI_105144;selecting a plant comprising the one marker nucleic acids, therebyselecting a maize plant having increased MRCV resistance; andpropagating the selected plant, thereby producing a plant that displaysincreased MRCV resistance.
 8. The method of claim 7, wherein at leasttwo marker nucleic acids are selected.
 9. The method of claim 7, whereinat least three marker nucleic acids are selected.
 10. The method ofclaim 7, wherein at least four marker nucleic acids are selected.